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Glenmark an Indian Pharmaceutical Company in Switzerland ndash Innovation
with a touch of spice
Sam Hou
Head Biologics Research Glenmark Pharmaceuticals SA
Mar 2014
1
bull Intro ndash Glenmark GBR 910 anti ndashTL1A antibody GBR830 anti-OX40 antibody BEAT bispecific antibody
2
Glenmark Pharmaceuticals - a research driven global integrated pharmaceutical company
bull Drug discovery business oriented towards out-licencing at various clinical stages
bull Branded generics business with a global presence
(EU US and across emerging markets including India)
3
Glenmark Pharmaceuticals
Turnover $32 MN 2 Formulations Manufacturing Facilities
Operations in 27 countries
Sales from International operations 8
Initiation into NCE research
2000
Turnover (FY 2012) $919 MN (+24)
12 Mfg Facilities over 4 Countries
Operations gt 95 Countries gt9000 employees
Sales from international operations gt70
Strong Pipeline of NCErsquos and NBErsquos
2013
API Mfg
Pure Generics Business
Branded Generics business
Proprietary Branded Business
O P E R A T I O N S A C R O S S T H E P H A R M A C E U T I C A L V A L U E C H A I N
4
Global Footprint
5
RampD for Biologics
Switzerland
Glenmark
Generics Ltd US
Glenmark
Generics SA
Argentina
Medicamenta
Czech Republic
Glenmark
Farmacetica Ltda
Brazil
Glenmark Pharma
Europe Limited
London UK
Bowter Bartlett
South Arica
Glenmark
India
Several other subsidiaries and representative offices exist across the world
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
bull Intro ndash Glenmark GBR 910 anti ndashTL1A antibody GBR830 anti-OX40 antibody BEAT bispecific antibody
2
Glenmark Pharmaceuticals - a research driven global integrated pharmaceutical company
bull Drug discovery business oriented towards out-licencing at various clinical stages
bull Branded generics business with a global presence
(EU US and across emerging markets including India)
3
Glenmark Pharmaceuticals
Turnover $32 MN 2 Formulations Manufacturing Facilities
Operations in 27 countries
Sales from International operations 8
Initiation into NCE research
2000
Turnover (FY 2012) $919 MN (+24)
12 Mfg Facilities over 4 Countries
Operations gt 95 Countries gt9000 employees
Sales from international operations gt70
Strong Pipeline of NCErsquos and NBErsquos
2013
API Mfg
Pure Generics Business
Branded Generics business
Proprietary Branded Business
O P E R A T I O N S A C R O S S T H E P H A R M A C E U T I C A L V A L U E C H A I N
4
Global Footprint
5
RampD for Biologics
Switzerland
Glenmark
Generics Ltd US
Glenmark
Generics SA
Argentina
Medicamenta
Czech Republic
Glenmark
Farmacetica Ltda
Brazil
Glenmark Pharma
Europe Limited
London UK
Bowter Bartlett
South Arica
Glenmark
India
Several other subsidiaries and representative offices exist across the world
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmark Pharmaceuticals - a research driven global integrated pharmaceutical company
bull Drug discovery business oriented towards out-licencing at various clinical stages
bull Branded generics business with a global presence
(EU US and across emerging markets including India)
3
Glenmark Pharmaceuticals
Turnover $32 MN 2 Formulations Manufacturing Facilities
Operations in 27 countries
Sales from International operations 8
Initiation into NCE research
2000
Turnover (FY 2012) $919 MN (+24)
12 Mfg Facilities over 4 Countries
Operations gt 95 Countries gt9000 employees
Sales from international operations gt70
Strong Pipeline of NCErsquos and NBErsquos
2013
API Mfg
Pure Generics Business
Branded Generics business
Proprietary Branded Business
O P E R A T I O N S A C R O S S T H E P H A R M A C E U T I C A L V A L U E C H A I N
4
Global Footprint
5
RampD for Biologics
Switzerland
Glenmark
Generics Ltd US
Glenmark
Generics SA
Argentina
Medicamenta
Czech Republic
Glenmark
Farmacetica Ltda
Brazil
Glenmark Pharma
Europe Limited
London UK
Bowter Bartlett
South Arica
Glenmark
India
Several other subsidiaries and representative offices exist across the world
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmark Pharmaceuticals
Turnover $32 MN 2 Formulations Manufacturing Facilities
Operations in 27 countries
Sales from International operations 8
Initiation into NCE research
2000
Turnover (FY 2012) $919 MN (+24)
12 Mfg Facilities over 4 Countries
Operations gt 95 Countries gt9000 employees
Sales from international operations gt70
Strong Pipeline of NCErsquos and NBErsquos
2013
API Mfg
Pure Generics Business
Branded Generics business
Proprietary Branded Business
O P E R A T I O N S A C R O S S T H E P H A R M A C E U T I C A L V A L U E C H A I N
4
Global Footprint
5
RampD for Biologics
Switzerland
Glenmark
Generics Ltd US
Glenmark
Generics SA
Argentina
Medicamenta
Czech Republic
Glenmark
Farmacetica Ltda
Brazil
Glenmark Pharma
Europe Limited
London UK
Bowter Bartlett
South Arica
Glenmark
India
Several other subsidiaries and representative offices exist across the world
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Global Footprint
5
RampD for Biologics
Switzerland
Glenmark
Generics Ltd US
Glenmark
Generics SA
Argentina
Medicamenta
Czech Republic
Glenmark
Farmacetica Ltda
Brazil
Glenmark Pharma
Europe Limited
London UK
Bowter Bartlett
South Arica
Glenmark
India
Several other subsidiaries and representative offices exist across the world
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Discovery Research Centres
350 Scientists based in Mumbai India Around 50 scientists based in Switzerland
6 Discovery
Candidates in
Clinical Trials
NCE Research NBE Research
Around USD 200 Million received from out-licensing deals of NCErsquos and NBErsquos
6
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Novel Drugs Pipeline Compound Primary Indications Target
Pre Clinicals Phasel Phase2 Phase3 Approval
HIV related Diarrhea CFTR Inhibitor
Adult Acute Infectious Diarrhea including Cholera
CFTR Inhibitor
Neuropathic Pain TRP A1 Inhibitor
Respiratory disorders TRP A1 Inhibitor
GRC 15300 Neuropathic Pain TRPV3 Antagonist
mPGES-1 inhibitors
Chronic Inflammatory conditions including Pain
mPGES-1 inhibitors
Vatelizumab Ulcerative Colitis VLA-2 Antagonist (mAb)
GBR 900 Chronic Pain TrkA Antagonists (mAb)
GBR 830 Autoimmune Disorders OX 40 Antagonist (mAb)
Out-licensed to Sanofi
In-licensed for ROW Markets
Option agreement with Forest Laboratories
In-licensed for ROW Markets
NCE
NBE
Crofelemer
GRC 17536
Out-licensed to Sanofi
7
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmark in Switzerland Glenmark Pharmaceutical Ltd (India) set up a wholly owned subsidiary Glenmark Pharmaceuticals
SA in Switzerland in July 2004
Biologics Research Lab established in 2006
Process Development established in 2008
GMP Manufacturing established in 2014
Team of 60 (Scientists ~ 50) focusing on developing monoclonal antibodies against inflammation and oncology disease areas
International team of highly qualified scientists with biopharmaceutical research experience Collaborations with numerous biologic companies amp leading academic institutions
8
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
RampD Biologics
Antibody Drug Discovery
Aspirin
Cyclosporine
9
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmarkrsquos GBR 910 TL1A Antagonist Program
10
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
TL1A (TNFSF15)
bull(discovered in 2002) is a member of the TNF ligand superfamily which comprises 18
different members in mammals
bull Type II transmembrane proteins homotrimeric Cleavable
- TL1A expression on
bullActivated Antigen-Presenting Cells (DC and macrophages)
bullActivated CD4+ and CD8+ T lymphocytes
bullTNFαIL-1 α activated endothelial cells
bull Binds to DR3 (death receptor 3 TNFRSF25 TRAMP LARD WSL-1) and DcR3 (decoy
receptor)
Migone 2002 Immunity 16 479-492
1-35 cytoplasmic 36-56 TM 57-251 extracellular
N C
11
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Biologics targeting TNF family members
12
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
TL1A linking innate and adaptive immunity
X
X
X
13
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
TL1A linking innate and adaptive immunity
14 Meylan Immuno Rev 2011
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
TL1A
Rheumatoid arthritis IBD
Asthma
Multiple sclerosis
(EAE)
Psoriasis
Potential role for TL1A the new TNF-family member and potent costimulator of IFN-gamma in mucosal inflammation
Prehn JL Mehdizadeh S Landers CJ Luo X Cha SC Wei P Targan SR
Expression Localization and Functional Activity of TL1A a Novel Th1-Polarizing Cytokine in Inflammatory Bowel Disease 1
Giorgos Bamias Charles Martin III Marco Marini Sharon Hoang Margarita Mishina William G Ross Muhammadreza A Sachedina Charles M
Friel
Role of TL1A in the Pathogenesis of Rheumatoid Arthritis1
Jun Zhang234Dagger Xuehai Wang2 Hassan Fahmi Susan WojcikDagger James FikesDagger Youhua Yusect Jiangping Wudagger and Hongyu Luo
Upregulation and nuclear localization of TNF-like cytokine 1A (TL1A) and its receptors DR3 and DcR3 in psoriatic skin lesions
Bamias G Evangelou K Vergou T Tsimaratou K Kaltsa G Antoniou C Kotsinas A Kim S Gorgoulis V Stratigos AJ Sfikakis PP
Essential role of TNF receptor superfamily25 (TNFRSF25) in the development of allergic lung inflammation (JEM 2008)
Lei Fang Becky Adkins Vadim Deyev and Eckhard R Podack
Selected
References
COPD
TL1ADR3 axis in inflammation and autoimmune diseases
atherosclerosis
15
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
bull TL1A-DR3 in health and disease
ndash High TL1A expression in synovial tissue of RA patients (membrane bound and soluble)
(Cassatella JI 2007 Bamias Clin Imm 2008Zhang JI 2009)
ndash High TL1A and DR3 expression in intestinal lamina propria of IBD patients (Bamias JI 2003Prehn Clin Imm 2004Kamada Inlamm
Bowel Dis 2009)
bull Mouse model of IBD (Takedatsu Gastroentero 2008)
16
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
TL1A in allergic lung inflammation (Fang JEM 2008)
Same observations were made in DR3-- animals (Meylan Immunity 2008)
0 5 12
-1 0 +1 +2 +3 +4
16
Ova+alum (ip) Ova+alum (ip) Ova (aero) Ab Ab Ab Ab Analysis
L4G6 blocking hamster anti-mouse TL1A mAb BALF Bronchoalveolar lavage fluid
mucus
In vitro stimulation of bronchial LN
Cytokine expression on lung parenchyma
Ab 50ug ip
Ab injected 4 hours before analysis
17
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Induction of TL1A expression in a mouse model of IBD
F480
F480
TL
1A
T
L1
A Naive mouse
Mouse with UC
UC= Ulcerative colitis
18
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Induction of TL1A expression in a mouse model of Allergic Asthma
Siglec F PE
CD
11c
AP
C
Siglec F-PE
Alveolar macrophages
Eosinophils
Isotype control on alveolar macrophages
TL1A on steady state alveolar macrophages
TL1A on asthmatic state alveolar macrophages
BAL of OVA-induced allergic asthma mouse
19
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
GBR910 blocks TL1A-induced IFN-γ secretion by primed CD4 T cells
IL-12 + IL18 - + + + + + +
IC monocytes - - + + + + +
5G6 (μgml) - - - 10 1 01 -
Isotype control - - - - - - +
Parental 5G6 candidate blocks
IFNg release induced by membrane bound TL1A
(produced by IC-activated monocytes)
IC Monocyte
Mb TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
Humanized 5G6 blocks IFNγ release induced
by soluble recombinant hTL1A
soluble TL1A
T cell
DR3
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
IFNg
20
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Number of eosinophils in BAL fluids
Contr
ol iso
Dex
amet
hasone
GBR91
0 (5
0mgk
g)
Nai
ve
00
2000000
4000000
6000000
8000000
nu
mb
er
of
eo
sin
op
hil
s
Her
ceptin
(20m
gkg)
Cyc
losp
orin
5G6
(20m
gkg)
No
DSS
5
6
7
8
colo
n l
eng
th (
cm
)
IBD and Allergic Asthma models
DSS colitis
OVA-induced asthma
21
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Day -1
Termination of the study
- Adhesion
- Strictures
- Ulcers
- Wall thickness
- HampE
- PAS
N=8
(rats Sprague Dawley)
Body weight
Day 0
Group1 Isotype control without TNBS
Group2 Isotype control+TNBS
Group3 GBR910
Group4 Prednisolone
starvation
+2 hours Day 7
treatment
Colon length
Histology
GBR910 in rat TNBS colitis model
22
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
00
05
10
15
20
25
30
35
Adhesions Strictures Ulcers score Thicknessscore
AV
ER
AG
E C
OLO
NIC
SC
OR
E
COLONIC PARAMETER
-TNBS +TNBS GBR910 prednisolone
plt005 by Students t-test
GBR910 ameliorates TNBS-induced inflamation in rat
23
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Summary Key attributes of GBR 910 and outlook
GBR 910 humanized mAb in IgG4HS backbone (blocker only)
Binding activity GBR 910 is cross-reactive with cyno sheep rat mouse
In vitro activity
- GBR 910 blocks binding of TL1A to DR3
-GBR 910 neutralizes IFNg-mediated T cell responses in vitro
In vivo GBR 910 ameliorates TNBS-induced inflammation in a rat model of Crohnrsquos disease (TNBS colitis)
Good anticipated safety profile Very narrow expression pattern
Envisaged other models of inflammatory disease in rat
COPD Chronic IBD models
24
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmarkrsquos GBR 830 OX40 Antagonist Program
25
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
OX 40 Target Characteristics Advantages in Targeting OX40
bull Co-stimulatory Receptor on T-cells tumor necrosis factor receptor superfamily member
bull Not found on naiumlve T-cells or memory T-cells transiently induced during T-cell activation
bull Specific for recently activated T-cells ndash No pan-immunosuppression
ndash Fewer severe infections and malignancies than small molecule and mAb immunosuppressants
26
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
OX40-OX40L Signalling in T-Cell Activation
27
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- OX40 co-stimulation only on activated T-cells after initial T-cell activation through CD28 - OX40-OX40L co-stimulation is a required step during clonal T-cell expansion and during memory cell reactivation
Survival Proliferation
Effector function Differentiation
OX40 signals OX40 signals
C D28 signals
memory memory
Naive
Effector
Expansion Contraction
Memory generation
Reactivation effector
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
OX40-OX40L Signalling Disruption in T-Cell Activation
28
OX40L (active homo-trimer)
OX40 (active homo-
trimer)
Antigen Presenting Cell
Activated T-Cell
- Disruption of OX40-OX40L interaction disturbs T-cell proliferation survival effector function and differentiation The net result is
- Weaker and shorter effector phase - Impaired memory generation and reactivation
- Early expansion of naiumlve T-cells not affected (allows limited T-cell effector function) innate immune responses are not affected by targeting OX40
Survival Proliferation
Effector function Differentiation
Smaller memory pool
Contraction sooner
Smaller effector Weaker late expansion
Weaker reactivation
Early expansion normal
memory memory Naive
effector
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
The OX40-OX40L Axis is well validated in Animal Models of Inflammatory and Autoimmune Disease
29 Croft (2010) 29
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Blocking OX40 does not prevent primary protective immune responses to acute infections Immunosuppressive effect observed only in chronic (memory) responses infections
Pathogen Mice or reagents Type of infection Response phenotype Reference
Leishmania major
OX40-- acute Normal clearance (C57BL6) Decreased pathology in BALBC J Immunol 1999 Dec 15163(12)6520-9
OX40L acute
Normal clearance (C57BL6) Decreased non protective TH2 response in BALBC J Exp Med 2000 Jan 17191(2)375-80
Nippostrongylus brasiliensis
OX40-- acute
Normal clearance Normal TH2 cytokines J Immunol 1999 Dec 15163(12)6520-9
Heligmosomoides polygyrus
OX40L-- chronic Increased infectivity Memory response altered J Immunol 2003 Jan 1170(1)384-
93
mCMV acute phase
OX40-- acute
Normal response
Weaker CD4 T cell response Normal CD8 and Ab response
J Immunol 2007 Aug 15179(4)2195-202
mCMV chronic phase
OX40--
chronic
Defective response Decreased long term CD8 and antibody response J Immunol 2007 Aug 15179(4)2195-202
VSV OX40-- OX40L--
acute
Normal clearance
Normal Ab response Reduced memory response
Immunity 1999 Dec11(6)699-708 Immunity 1999 Dec11(6)689-98
Theilers murine encephalomyelitis virus
OX40-- acute
Normal clearance Normal CD8 response J Immunol 1999 Dec 15163(12)6520-9
Influenza OX40-- OX40-Ig
acute
Normal clearance
Reduced pulmonary CD4 and CD8 normal Ab response Reduced immunopathology
Immunity 1999 Dec11(6)699-708 J Immunol 2005 Aug 1175(3)1665-76 J Exp Med 2003 Oct 20198(8)1237-42
Acute LCMV OX40-- acute
Normal response Normal primary and memory CD8 response Decreased CD4 response Normal Ab response
Immunity 1999 Dec11(6)699-708
Persistent LCMV OX40-- chronic
Defective response
Decrease initial pathology but impaired anti viral T cell accumulation
PLoS Pathog 2012 Sep8(9
Mycobacterium avium
OX40L acute
Normal clearance
J Leukoc Biol 2004 Nov76(5)1039-46
Listeria monocytogenes OX40-- acute
Normal primary response and clearance
Normal primary CD8 Decreased long term and memory CD8 J Immunol 2008 Nov 1181(9)5990-6001
30
ProtozoaNematode
Virus
Bacteria
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Translational Validation of OX40-OX40L Axis in Human Disease States
31
bull Tissue Expression ndash Psoriasis (Matsumura Arch Dermatol res 2003)
ndash Atopic dermatitis (Ilves J Eur Acad Dermatol Venereol 2013)
ndash RA (Giacomelli Clin Exp Rheumatol 2001 Brugnoni Br J Rheumatol 1998)
ndash IBD (Souza Gut 1999 Stuumlber Eur J Clin Invest 2000)
ndash Lupus (Dolff Arthritis Res Ther 2010 Aten J J Am Soc Nephrol 2000 Patschan S Clin Exp Immunol 2006)
Atopic dermatitis
Psoriasis
Healthy control
Healthy control
OX40 immunohistology
Lupus nephritis
Ilves et al
Dolff et al
Souza et al
Patschan et al
SLE
IBD
CD3+ OX40+ CD4+ OX40+
SF Synovial fluid PB peripheral blood
Giacomelli et al
OX40 flow cytometry
Colon
Peripheral blood
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
GBR 830 Blocks OX40OX40L Interaction and has cytotoxic potential
32
ELISA anti BrdU Readout cell proliferation
ADCC on HPB-ALL (OX40+ T lymphoma line)
OX
40
L
OX
40
T
C
R
OX
40
LOKT3
T-cell
OX
40
-Fc
OX
40
-Fc
OX
40
-Fc
OX
40
L
OX
40
L
OX
40
L
-4 -3 -2 -1 0 1 200
05
10
Non blocker anti OX40
GBR 830
No Ab
Log antibody concentration (gmL)
OD
(450n
m)
10 10 1 01 001
0
50
100
Isotype control
GBR 830
Antibody concentration (gmL)
Inh
ibit
ion
of
pro
life
rati
on
(
)
-8 -6 -4 -2 0 2 4-10
-5
0
5
10
15
20IgG1 control
GBR 830
GBR 830-IgG4hs
Log antibody concentration (gmL)
Sp
ecif
ic A
DC
C (
)
Readout Killing
NK cell
T-cell
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
OX40 Antagonist Inhibits Mixed Lymphocyte Reaction
33
Legend One way mixed lymphocyte reaction (MLR) measured by 3H thymidine incorporation Bars show the mean 3H -thymidine incorporation (counts) of at least triplicates plusmn standard error of the mean Isotype control (trastuzumab) and positive control (efalizumab) are shown Effector stands for only effector cells Effector + target represents a measurement where antibodies have been omitted
bull In vitro validation of inhibition of T-cell proliferation by OX40 antagonist mAb bull Potency on parsuperior to Efalizumab (LFA1 blocker)
effe
ctor
effe
ctor+
targ
et
Isoty
pe co
ntrol (
50ugm
L)
Efaliz
umab
(20u
gml)
Efaliz
umab
(2ugm
l)
Efaliz
umab
(00
2ugm
l)
(50u
gml)
(5ugm
l)
(05
ml)
(00
5ugm
l)
(00
1ugm
l)
(00
01ugm
l)
0
2000
4000
6000
3H
in
co
po
rati
on
(re
lati
ve p
roli
fera
tio
n)
OX40 antagonist
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Xenogeneic human graft versus host (mouse) reaction
34
Legend Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million human PBMCs intraperitoneally The mice were also depleted for mouse NK cells by twice weekly injections with the TMbeta1 antibody The treatment with OX40 antagonistic antibody (1 or 10mgkg) Etanercept (a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 Amgen-Pfizer ) or vehicle was given iv weekly for six consecutive doses and started two days before the PBMC injection The animals were checked and scored three times weekly for GVHD symptoms including weight loss diarrhea fur aspect and general behavior Animals were ethically sacrificed if symptoms were considered too severe
Treatment
0 20 40 600
50
100 Vehicle
Enbrel (8mgkg)
OX40 antagonist 1mgkg
OX40 antagonist 10mgkg
days
Perc
en
t su
rviv
al
bull In vivo validation of inhibition of T-cell proliferation and effector function by OX40 antagonist mAb bull Potency superior to Etanercept
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
GBR 830 displays a potent therapeutic anti-psoriasis effect
35
bull Relevance of the psoriasis plaque transplant model
bull Method Overview ndash Lesional psoriatic patient skin (epiderma+derma) patches are grafted on a SCID mouse
After graft implementation the mice are treated Main readout epidermal thickness and T cell infiltration
bull Advantages ndash Readouts are clinically relevant
ndash Genuine pathologic human tissue
ndash Healing process in grafted tissue due to treatment is very similar to healing of a normal tissue in a patient
ndash Pathology is dependent on leukocytes (mostly activated memory T cells) in the transplant
bull This model is highly predictive of clinical efficacy ndash Therapeutics that have successfully been tested in this model CsA TNF-α blockers
(infliximab etanercept) ustekinumab (anti IL-12IL-23) Anti IL-17 Anti CD11a (raptiva) Anti IL-21)
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Effect on psoriasis healing
36
bull Treatment with GBR 830 improved resolution of psoriasis phenotype
bull Same effect observed on all the 5 donors included bull Indicates that targeting OX40 can impact an ongoing
chronic (memory) pathological response
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Donor 1-5
Isoty
pe co
ntrol
GBR83
0
Temova
te
0
50
100
150
200
250
Ep
iderm
al
thic
kn
ess (
m)
Donor 1 Donor 4
Iso
typ
e
con
tro
l G
BR
83
0
Tem
ova
te
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmarkrsquos BEATreg Technology
Bispecific Antibody Platform
37
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific at Scale
bull Plug and play from existing binders bull Effector functions preserved bull Low mispairing of binders
bull Stable high expression
bull Utilization of standard methods and equipment bull High yield
38
Bispecific Engagement by Antibody based on T-cell receptor
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
BEATreg technology based on bio-mimicry
39
Engineering of CH3 interface heavy chain heterodimerization technology mimics the natural association of the T-cell surface receptors α and β
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Glenmarkrsquos BEATreg Technology
Bispecific Antibody
Engineering Platform
Cell-Line Development and
Master-Cell Banking
Manufacturing of Bispecific molecules
bull Nature-like heterodimeric molecule bull Full antibody Fc functions incl half life
bull Industrial relevant expression in CHO cells
bull gt 3gL bull gt 90 heterodimer bull Stable product quality
bull Industrial relevant scalable purification process
bull gt 97 of heterodimer after Protein A
40
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
T cell-mediated redirected killing - a novel BEATreg mechanism of action
Bridging T cells to tumor cells via anti CD3tumor antigen bispecific molecule allows activation of T cells independently of tumor peptide recognition Leads to redirected lysis (RDL) and death of tumor cell
CD3+ T cell
HER2+ Tumour Cell
41
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
BEATreg amp CD3 Redirected Killing
bull CD3 x Her2 efficiently kills receptor expressing
cell line
bull picomolar killing activity of BEAT
Legend Effector cells human T-cells Target cells expressing Her2 receptor EffectorTarget = 51 24h incubation readout FACS 7-AADCFSE
42
0
20
40
60
80
100
No Ab
Ctrl BEAT
CD3 x Her2
Concentration (pM)
o
f s
pec
ific
kil
lin
g
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
CD3 x Her-2 bispecific ndash Her2+ SCID mouse xenograft in vivo model MOA redirected killing JIMT-1 human breast adenocarcinoma ndash resistant to herceptin
Average of tumor growth volume for all mice in all groups
BEATreg amp CD3 Redirected Killing
43
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
44
PUBLISHED APPLICATIONS WO2013183032 HUMANIZED ANTI-TRKA ANTIBODIES WITH AMINO ACID SUBSTITUTIONS WO2013084157 EXPRESSION CASSETTE WO2013008171 ANTIBODIES THAT BIND TO OX40 AND THEIR USES WO2012131555 HETERO-DIMERIC IMMUNOGLOBULINS WO2010095031 HUMANIZED ANTIBODIES THAT BIND TO CD19 AND THEIR USES WO2010052556 TREATMENT WITH ANTI-ALPHA2 INTEGRIN ANTIBODIES WO2009093138 HUMANIZED ANTIBODIES SPECIFIC FOR VON WILLEBRAND FACTOR WO2007056858 ANTI-ALPHA2 INTEGRIN ANTIBODIES AND THEIR USES FILED APPLICATIONS TO BE PUBLISHED PCTEP2013069989 PURIFICATION OF HETERO-DIMERIC IMMUNOGLOBULINS (Pub date scheduled Mar 2014) PCT XXXX ANTIBODIES THAT BIND TO TL1A AND THEIR USES (Pub date scheduled Jun 2014) USXXXX ldquoGBR 200 FORMULATIONrdquo PCTEPXXXX ALTERNATIVE SPLICING
Intellectual Property
45
Thank You
Recommended